The present invention relates to a biochemical methods, and kits to measure the niacin status of an individual, particularly to a colorimetric method for the detection of intracellular pyridine nucleotide content or concentration in a biological sample such as whole blood or tissues.
Niacin, also called nicotinic acid, pyridine 3-carboxylic acid, vitamin B3 or vitamin PP, is a water soluble vitamin. The known biological roles of niacin are attributable to the function of its active metabolites, nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP).1 As used herein, pyridine coenzymes or pyridine nucleotides refer to NAD and NADP. NAD and NADP represent the total oxidized and reduced pools of each nucleotide, respectively. Thus, NAD represents the sum of NAD+ and NADH while NADP represents the sum of NADP+ and NADPH.
1xe2x80x2 NAD is also called diphosphopyridine nucleotide (DNP) and conenzyme I or cozymase. NADP is also called triphosphopyridine nucleotide (TPN) or coenzyme II. These alternative names are not popular or are no longer used. 
In humans, niacin equivalents can be obtained from dietary nicotinate, nicotinamide, and tryptophan. Consequently, niacin status depends on the amount of these in the diet and on factors that influence uptake, distribution, efficiency of conversion to tissue NAD and NADP, and excretion or reutilization of the nicotinamide moiety formed by the turnover of NAD and NADP. The term niacin number has been chosen as a convenient way to represent niacin status and is defined as the ratio of the concentration of NAD to the concentration of NADP multiplied by 100 (100xc2x7[NAD]/[NADP]) in whole blood. For example, if the relative concentration of NADP([NADP]) is 1 and the relative concentration of NAD([NAD]) is 0.72, the niacin number would be 72 (from the formula 100xc2x7[NAD]/[NADP]=72 wherein [NAD] is 0.72 and [NADP] is 1). Expressing the values in this way yields a whole number that is linearly related to intracellular NAD content of red blood cells.
Attempts have been made to measure niacin status in a subject. Previous attempts have involved the determination of urinary metabolites of nicotinamide (R. A. Jacob et al., J. Nutr. 119, 591 (1989)). In these tests the urinary excretion of niacin or niacin metabolites are measured to infer the niacin status in a subject. These attempts involve multiple biochemical steps that are labor intensive, and slow and do not measure niacin bioavailability or intracellular niacin active metabolites directly. Thus, current methods for measuring niacin status are not suitable to wide-scale screening and the relationship of the nicotinamide metabolites to niacin status is still poorly understood.
A metabolic ward study has shown that the NAD content of erythrocytes is a sensitive marker for niacin status in humans (C. S. Fu et al., J. Nutr. 119, 1949 (1989)). Test subjects restricted to a niacin intake of approximately 50% of the recommended daily allowance showed a 70% decrease in NAD content after five weeks. In contrast, NADP content remains relatively constant throughout the niacin restricted diet period (C. S. Fu et al., J. Nutr. 119, 1949 (1989)). Thus, because the NADP concentration remains constant while the NAD concentration is affected by niacin intake, the ratio of NAD to NADP reflects niacin status. Niacin status refers to the bioavailability of niacin and niacin derivatives such as NAD and NADP inside a cell and is an indication of the bioavailability of NAD. Because more than 98% of the total pyridine nucleotide pool of whole blood is in the erythrocyte fraction, (E. L. Jacobson and M. K. Jacobson, J. Int. Med. 233, 59 (1993)) niacin status can be obtained from a few microliters of whole blood. One aspect of the invention is directed to a method of assessment of niacin status which is inexpensive, relatively accurate and rapid, and suitable to wide scale-application in the human population.
Niacin status derived from erythrocytes or whole blood from humans varies over a wide range. The data of Table I show the mean niacin number and the range of values measured in several populations. Using data from a nonrandom population of free living healthy adults and metabolic ward subjects on controlled niacin intake (C. S. Fu et al., J. Nutr. 119, 1949 (1989)), the mean niacin number is found to be approximately 175 and from the standard deviation it is predicted that 95% of the population would have values between 127 and 223. In a separate study of a large population of 46- to 64-year-old individuals in Malmxc3x6, Sweden a range of 28 to 337 was seen, with a mean of 160. The effect of dietary niacin intake on niacin status was shown in a study of individuals undergoing niacin therapy where the average pretherapy value of 175 was increased to 665 by niacin supplements. Taken together these data illustrate that niacin status varies widely in the human population and can be modulated by niacin supplementation.
The assay of the invention is useful for determining the optimal amounts of dietary niacin to obtain an optimal level of intracellular niacin metabolites (niacin number). NAD is involved with ADP-ribose transfer reactions and these reactions have been implicated in a number of metabolic signaling processes (M. K. Jacobson, et al., in ADP-Ribosylating Toxins and G Proteins: Insights into Signal Transduction, J. Moss and M. Vaughan, eds., p. 479 American Society for Microbiology, Washington, DC 1990; K. C. Williamson and J. Moss, in ADP-Ribosylating Toxins and G Proteins: Insights into Signal Transduction, J. Moss and M. Vaughan, eds., p. 493. American Society for Microbiology, Washington, D.C., 1990; M. A. De Matteis et al., Proc. Natl. Acad. Sci. U.S.A. 91, 1114 (1994); H. C. Lee et al., Vitam. Horm. 48, 199 (1994); F.-J. Zhang et al., Bioorg. Med. Chem. Lett. 5,2267 (1995); C. Q. Vu et al., J. Biol. Chem. 271, 4747 (1996)) and in cellular recovery from DNA damage (F. R. Althaus and C. Richter, xe2x80x9cADP-Ribosylation of Proteins: Enzymology and Biological Significance.xe2x80x9d Springer-Verlag, Berlin, 1987). For example, studies of the conversion of NAD to ADP-ribose polymers in response to DNA damage indicate that an optimal cellular content of NAD may be a preventive factor in cancer (E. L. Jacobson and M. K. Jacobson, J. Int. Med. 233, 59 (1993); E. L. Jacobson et al., in ADP-Ribosylating Reactions, G. G. Poirier and P. Moreau, eds., p. 153. Springer-Verlag, New York, 1992). Such studies have shown a need for a rapid and accurate method for measuring niacin content based on tissue NAD in humans.
There is a need for a highly sensitive, accurate and reliable method to determine niacin status as measured by intracellular NAD content relative to the intracellular NADP content in a subject in order to conveniently and rapidly assay the niacin state in a subject. Such a method should be inexpensive, easy to manufacture as a kit, easy to use, adaptable to current laboratory equipment, and be capable of miniaturization and automation.
The present invention is directed to meeting the foregoing needs by providing a simple and convenient method of assaying intracellular pyridine nucleotide in a subject. The subject may be a mammal, such as, for example, a human being. The invention can be applied to the field of medicine where measurement of niacin is undertaken in diagnosis involving human subjects.
One advantage of the method of the invention is that it has high sensitivity and a wide range of linearity, thus enabling the direct and rapid measurement of pyridine nucleotides in biological fluids containing cells such as blood without sample concentration or dilution.
Interest in measuring niacin and pyridine nucleotide in humans has increased since it was demonstrated that total pyridine nucleotide concentrations reflect niacin intake in humans and that niacin plays a role in prevention of cancer. Prior art methods of niacin measurement require expensive, laborious analyses of oxidized products of niacin in urine or even more tedious assays of nicotinamide and/or nicotinic acid in serum. The current methods are disadvantageous at least because they do not measure the intracellular active forms of niacin, NAD and NADP. Thus, a need has arisen for a simple and effective way to measure niacin status.
This need has been met by the present invention. The present invention involves a novel double extraction procedure that can allow quantification of total pyridine nucleotides from blood cells from samples as small as 10 xcexcL of whole blood. The present invention avoids the above noted disadvantages of prior art niacin measurement methods and provides unique advantages over all prior approaches to assessing niacin status in humans. One advantage of the method is that it is capable of directly measuring intracellular NAD and NADP. Because the measurement is extremely sensitive, it can be made using a very small amount of blood. The 10 xcexcL of blood required for the assay is typically available as a residual amount of blood drawn for other clinical measurements.
Another advantage of the method is that pyridine nucleotides are assayed directly by the addition of a few solutions with no additional fractionation, concentration, or dilution steps. Further, it has been demonstrated that the extraction procedure results in the complete extraction of total pyridine nucleotides (NAD and NADP) without any losses of either the oxidized or reduced forms of the nucleotides. Another advantage of the method is that the amount of manual labor to perform each assay is minimal and thus highly adaptable to automated or semiautomated operation. For example, a skilled operator using a multichannel pipette (Sigma, St. Louis) with (8, 12 or 96 channels) may perform assays using one (96 assays), two (192 assays), three (288 assays) or four (384 assays) or more microtiter dishes simultaneously. For example, the centrifugation steps of the method may be performed directly on a microtiter dish using a microtiter dish centrifuges (Beckman, Fullerton, Calif.).
The methods of the invention may be used in the context of health maintenance and disease prevention. Given the role of niacin in the biological responses of a cell to carcinogenic insults, the niacin status of an individual may be a critical factor in determining the overall ability of an individual to withstand such insults. The simplicity of the method and the small amount of sample required make it particularly well suited for adaption to a home test kit for niacin status. In an embodiment, a home test kit may comprise a lancet and a tube for blood. The user simply need to draw about 10 xcexcL to 100 xcexcL of blood, mix the blood with anticoagulant, and send the sample to the laboratory for analysis. The anticoagulation step may be performed by adding a small amount of anticoagulant to the collected blood. Alternatively, to simplify the collection process, the anticoagulants may be incorporated into the collection tube, such as, for example, as a dry coating in a collection kit for the consumer.
The invention is now described in the following specific embodiments:
Assaying Intracellular Pyridine Nucleotide
One embodiment of the invention is directed to a method of assaying an intracellular pyridine nucleotide status of a biological sample. The method comprises obtaining the biological sample and extracting the intracellular pyridine nucleotide from the biological sample. The amount of pyridine nucleotide in the sample is detected in a cycling process which is summarized in FIG. 1. Briefly, the cycling process contains the following two steps which can be performed in any order. In the first step, the pyridine nucleotide is reduced to a reduced pyridine nucleotide. In the second step, an electron from the reduced pyridine nucleotide is transferred to an electron acceptor dye molecule. The transfer of an electron from the reduced pyridine nucleotide to the electron acceptor dye compound causes the reduced pyridine nucleotide to become oxidized. Thus, after this second step, the first step may be performed to reduce the pyridine nucleotide again.
It is understood that in some instances, the extracted pyridine nucleotide is already reduced and thus the reaction is started by step 2 first (transfer electron to dye) followed by step 1 (reduce pyridine nucleotide). It is further understood that both steps may be performed in any particular order and preferably the two steps are performed simultaneously. That is the reduction of pyridine nucleotide may be performed in the same reaction where an electron from the reduced nucleotide is transferred to the electron acceptor dye. For example, an enzyme and enzyme substrate pair (discussed in a later section) may be added to reduce pyridine nucleotide at the same time a electron acceptor dye and an electron transmitter compound (discussed in a later section) is added to remove electron from the pyridine nucleotide.
The result of the cyclic reaction is that an electron is transferred to an electron acceptor dye (the dye is reduced) at a rate that is proportional to the amount of pyridine nucleotide present in the sample. The reduction of the electron acceptor dye is measured to determine the amount of pyridine nucleotide in the biological sample. The reduction of the electron acceptor dye may be measured by a shift in absorbance of the dye in response to the accepted electron. The exact amount of pyridine nucleotide present may be determined by first performing the method of the invention with different known amounts of pyridine nucleotide and recording the reduction of the electron acceptor dye in response to each quantity of pyridine nucleotide. A correlation curve may be drawn to show the correlation between the pyridine nucleotide present and the shift in absorbance of the dye (amount of electron acceptor dye reduction). Then the method is used to determine the pyridine nucleotide of an unknown sample. The amount of shift in absorbance is compared to the correlation curve to determine the amount of pyridine nucleotide in the unknown sample.
One advantage of the method is that it is highly sensitive. The cycling reaction allow one pyridine nucleotide to facilitate the transfer of multiple electrons to an electron acceptor dye. In this way the signal is amplified. Further, the reaction proceeds linearly with time such that the reaction time may be lengthened to increase the sensitivity. The pyridine nucleotides (NAD and NADP) is stable for at least 1.5 hours. Thus, the detecting step may be, for example, about one minute, about 3 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 60 minutes, about 90 minutes, about 120 minutes or longer or any value of time in between.
The transfer of an electron to the electron acceptor dye causes a change in the absorbance of the dye. This change may be measured by eye. More preferably, the change in absorbance is measured by a photospectrometer. Depending on the electron acceptor dye used, the change may be in the visible, infra red, or ultraviolet region of the spectrum.
The pyridine nucleotide to be measured may be NAD or NADP. It is understood that NAD refers to the reduced form (NAD+) and the oxidized form (NADH). It is understood that NADP refers to the reduced form (NADPxe2x88x92) and the oxidized form (NADPH).
The Biological Sample
The biological sample may be whole blood or a solid tissue obtained from a subject. The subject may be any animal. Preferably, the animal is a mammal. Even more preferably, the mammal is a human. The biological sample may be any tissue from the subject that comprises whole cells. One advantage of the method of the invention is that an intracellular niacin status may be determine from whole blood. If the assay is performed immediately, whole blood may be used directly. If a period of time, such as a few minutes is to pass between the collection of the sample and the assay, then the whole blood should be anticogulated.
The extraction step comprises disrupting the cells in the biological sample and removing proteins from the disrupted cells. In a preferred embodiment, disruption is performed by treating the biological sample with a basic compound for a period of less than about 2 minutes at less than about 4xc2x0 C. followed by neutralizing the basic compound. Alternatively, a frozen biological sample may first be disrupted by mechanical means in liquid nitrogen before treatment with a basic compound and neutralization. Protein removal may be performed by an acid precipitation or salt precipitation of proteins. Acid precipitation may involve acid precipitation of proteins followed by neutralization of the acid.
A Cycling Reaction to Quantitate Pyridine Nucleotide
The amount of pyridine nucleotide in the extract is quantitated by a two step cycling reaction. In one step of the cycling reaction, pyridine nucleotide is reduced. Reduction may be performed by adding an enzyme and enzyme substrate pair to the extracted pyridine nucleotide. For the measurement of NAD, the enzyme and enzyme substrate pair may be alcohol dehydrogenase and ethanol; malate dehydrogenase and malate; lactate dehydrogenase and lactate; cytoplasmic isoctirate dehydrogenase (cytoplasmic) and isocitrate; glyceraldehyde-3-phosphate dehydrogenase and glyceraldehyde-3-phosphate; or a combination of these enzyme and enzyme substrate pairs. For the measurement of NADP, the enzyme and enzyme substrate pair may be mitochondrial isocitrate dehydrogenase and isocitrate; glucose-6-phosphate dehydrogenase and glucose-6-phosphate; 6-phosphogluconate dehydrogenase and 6-phosphogluconate; malic enzyme and malate; mitochondrial isocitrate dehydrogenase and isocitrate; or a combination of these enzyme and enzyme substrate pairs. After reduction, the pryidine compounds NAD and NADH should be reduced to NADH only. Similarly, the pryidine compounds NADP and NADPH should be reduced to NADPH only. The method is not limited to any particular enzyme and enzyme substrate pair. Any enzyme and enzyme substrate capable of reducing NAD and NADP may be used. Other NAD specific or NADP specific enzymes may be determined by consulting standard biochemical references. Such pairs may include artificial or genetically engineered enzymes and substrates. In addition, total pyridine nucleotide (NAD and NADP) may be measured together by adding NAD and NADP specific enzyme. Naturally, because the total pyridine nucleotide concentration is equal to the sum of the NAD concentration and NADP concentration, NAD concentration is equal to total pyridine concentration minus NADP concentration. Similarly, NADP concentration is equal to total pyridine concentration minus NAD concentration.
In the second step of the cycling reaction, an electron is transferred from the reduced pyridine nucleotide to an electron acceptor dye. An electron may be transferred from the reduced pyridine nucleotides (NADH or NADPH) to an electron acceptor dye via intermediate electron transmitter compound. Any electron transmitter compound capable of accepting an electron from a reduced pyridine nucleotide and transferring an electron to an electron acceptor dye molecule may be used. Preferred electron transmitter compound include, for example, oxidized phenazine ethosulfate (PES(ox)), 5-methylphenazinium methylsulfate, 1-methoxy-5-methylphenazinium methylsulfate, diaphorase (dihydrolipoamide reductase, EC 1.6.4.3.) or a combination of these electron transfer compounds. In the transfer reaction, the electron from NADH or NADPH is first transferred to an electron transmitter compound, then an electron from electron transfer compound is transferred to an electron acceptor dye molecule.
After the second step of the cycling reaction, the pyridine nucleotide is oxidized again. That is NADH is converted to NAD and NADPH is converted to NADP. The oxidized pyridine nucleotide may serve as substrate for the first step of the cycling reactionxe2x80x94the reduction of pyridine nucleotide. The cycle may be repeated many time to generate a detectable signal from a small concentration of pyridine nicleotide.
The Electron Acceptor Dye
The electron acceptor dye molecule may be any dye molecule that shows a detectable absorbance change after acceptance of an electron. Preferred electron acceptor dye compound include, for example, thiazolyl blue (MTT), 3,3xe2x80x2-(3,3xe2x80x2-dimethoxy-4,4xe2x80x2-biphenylene)-bis(2-(p-nitrophenyl)-2H-tetrazolium chloride) (NBT), 3-(p-indophenyl)-2-(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT), 3,3xe2x80x2-(4,4xe2x80x2-biphenylene)-bis(2,5-diphenyl-2H-tetrazoliumchloride), 3,3xe2x80x2-(3,3xe2x80x2-dimethoxy-4,4xe2x80x2-biphenylene)-bis(2,5-diphenyl-2H-tetrazolium chloride), 3,3xe2x80x2-(3,3xe2x80x2-bis(2,5-bis(p-nitrophenyl)-2H-tetrazolium chloride) and a combination of these dyes.
Calculation of Niacin Status
Another embodiment of the invention is directed to a method of calculating a niacin status in a biological sample. A niacin status may be express as niacin number=(100xc2x7[NAD]/[NADP]) or expressed as a ratio of NAD to total nucleic acid, NAD to total protein, NADP to total nucleic acid or NADP to total protein. To calculate a niacin number, any method of the invention is used to determine the amount of NAD in a biological sample. Any method of the invention is used to determine the amount of NADP in a biological sample. The niacin status, expressed as a niacin number, may be derived from the formula: niacin number=(100xc2x7[NAD]/[NADP]).
Alternatively, it is possible to determine the ratio of pyridine nucleotide to total cellular nucleic acid (DNA, RNA or DNA+RNA) or to total cellular protein. The pyridine nucleotide, which may be the amount of NAD or the amount of NADP is measured using any of the methods of the invention. Total cellular DNA or total cellular protein may be measured using standard methods. A ratio of pyridine nucleotide to total cellular DNA or protein can be calculated from the results.
Measuring Niacin Status from Whole Blood
Another embodiment of the invention is directed to a method to measure intracellular niacin status in whole blood. In the method, a whole blood sample is collected. Intracellular pyridine nucleotide is extracted from the whole blood sample. A cycling reaction, as discussed above, is used to transfer an electron to an electron acceptor dye. After the cycling process, the change in absorbance of the electron acceptor dye molecule is measured to determine the intracellular pyridine nucleotide status. The method may be performed immediately after collection of whole blood. Alternatively, if the method cannot be performed immediately, the whole blood may be anticogulated.
Measuring Reduced Intracellular Pyridine Nucleotide
Another embodiment of the invention is directed to a method for measuring a reduced intracellular pyridine nucleotide in a biological sample. In the method, a biological sample is provided. The reduced intracellular pyridine nucleotide from the biological sample is extracted with hot alkaline treatment as described in the Examples section. Briefly, the sample is placed in ice-cold 1.0 M NaOH extract and then heated with stirring at 60xc2x0 C. for 10 min. The amount of NaOH used may be, for example, about 0.1 volume, about 0.3 volume, about 0.5 volume, about 1 volume, about 3 volume, about 5 volume or about 10 volume of the biological sample. After the hot alkaline treatment, the sample is neutralized, and chilled. The heating destroys oxidized nucleotides and the resulting extract is a measure of the reduced pyridine nucleotides. The reduced intracellular pyridine nucleotide is used to mediated the transfer of an electron to an electron acceptor dye using the cycling reaction as described in the section entitled xe2x80x9cA Cycling Reaction to Quantitate Pyridine Nucleotide.xe2x80x9d The change in absorbance of the electron acceptor dye molecule is measured to determine the amount of intracellular reduced pyridine nucleotide.
Measuring Total Intracellular Pyridine Nucleotide
Another embodiment of the invention is directed to a method for measuring a total intracellular pyridine nucleotide in a biological sample. In the method, a biological sample is provided. The reduced intracellular pyridine nucleotide from the biological sample is extracted with cold acid treatment as described in the Examples section. Briefly, the sample is placed in ice-cold 1.0 M NaOH extract. The sample is neutralized immediately with ice-cold H3PO4. Neutralization refers to the addition of acid, base or a buffer to a solution until the pH is between about 5 and about 10, preferably between about 6 and about 8, more preferably between about 6.5 to 7.5 and most preferably about 7. The extracted total intracellular pyridine nucleotide is used to mediated the transfer of an electron to an electron acceptor dye using the cycling reaction as described in the section entitled xe2x80x9cA Cycling Reaction to Quantitate Pyridine Nucleotide.xe2x80x9d The change in absorbance of the electron acceptor dye molecule is measured to determine the amount of intracellular total pyridine nucleotide.
Kits
Another embodiment of the invention is directed to a kit for the extraction of intracellular pyridine nucleotides from a biological sample. The kit may comprise a base solution, an acid solution, a neutralizing solution, and an electron acceptor dye. The base solution may be NaOH, the acid solution may be HClO4 and the neutralizing solution may be KOH. The electron acceptor dye may be any dye that can accept an electron from NAD, NADP or an electron transmitter compound. Examples of preferred dyes include thiazolyl blue (MTT); 3,3xe2x80x2-(3,3xe2x80x2-dimethoxy-4,4xe2x80x2-biphenylene)-bis(2-(p-nitrophenyl)-2H-tetrazolium chloride) (NBT); 3-(p-indophenyl)-2-(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT); 3,3xe2x80x2-(4,4xe2x80x2-biphenylene)-bis(2,5-diphenyl-2H-tetrazoliumchloride); 3,3xe2x80x2-(3,3xe2x80x2-dimethoxy-4,4xe2x80x2-biphenylene)-bis(2,5-diphenyl-2H-tetrazolium chloride); 3,3xe2x80x2-(3,3xe2x80x2-bis(2,5-bis(p-nitrophenyl)-2H-tetrazolium chloride); and a combination thereof When multiple dyes are used in a reaction, the absorbance of each dye may be measured individually.
The kit may optionally comprise one or more of the following: an electron transfer compound, an enzyme and enzyme substrate pair, NAD or NADP pyridine nucleotide standard, and instructions for performing an intracellular pyridine nicleotide assay using any of the methods of the invention.
The electron transfer compound of the kit may be any compound capable of accepting an electron from a reduced pyridine nucleotide and transferring an electron to an electron accentor dye molecule. Examples of electron transmitter compound include oxidized phenazine ethosulfate (PES(ox)); 5-methylphenazinium methylsulfate; 1-methoxy-5-methylphenazinium methylsulfate; diaphorase (dihydrolipoamide reductase, EC 1.6.4.3.); and a combination of these compounds.
The enzyme and enzyme substrate pair of the kit may be any enzyme and enzyme substrate that together can cause a reduction of NAD or NADP. In a kit or a reaction designed to measure NAD, the enzyme and enzyme substrate should be specific for reducing NAD. Similarily, in a kit or a reaction designed to measure NADP, the enzyme and enzyme substrate should be specific for reducing NADP. If both NAD and NADP is measured in one reaction, the enzyme and enzyme substrate need not be specific for reducing NAD or NADP but can reduce both pyridine nucleotides. Examples of preferred enzyme and enzyme substrates include alcohol dehydrogenase; ethanol; malate dehydrogenase; malate; lactate dehydrogenase; lactate; NAD specific isoctirate dehydrogenase; isocitrate; glyceraldehyde-3-phosphate dehydrogenase; glyceraldehyde-3-phosphate; glucose-6-phosphate dehydrogenase; glucose-6-phosphate; 6-phosphogluconate dehydrogenase; 6-phosphogluconate; malic enzyme; malate; NADP specific isocitrate dehydrogenase; and a combination thereof.